Many physiologically active peptides, such as calcitonin, growth hormone releasing factor, LH-RH (luteinizing hormone releasing hormone), vasopressin, gastrin, .alpha.-MSH (.alpha.-melanotropin) and the like, are active only if their C-terminus is amidated. However, it is difficult to industrially produce the C-terminally amidated peptides directly by a chemical synthesis or a direct genetic engineering process. Their production can be better achieved by a two-step process in which in the first step a non-amidated peptide is produced which is in the second step amidated at its C-terminus. An example of such a process comprises synthesizing a peptide having an additional glycine unit on its C-terminus and converting it in the second step to a C-terminally amidated peptide using a C-terminally amidating enzyme, i.e. a peptidylglycine .alpha.-amidating monooxygenase (PAM).
C-terminally amidating enzymes (PAMs) of various origins were purified and corresponding cDNAs were prepared, such as of Xenopus laevis skin (Mizuno, K. et al., Biochem. Biophys. Res. Commun. 137, 984-991, 1986; Mizuno, K. et al., Biochem. Biophys. Res. Commun. 148, 546-553, 1987; Ohsuye, K. et al., Biochem. Biophys. Res. Commun. 150, 1275-1281, 1988), porcine pituitary gland (Kizer, J. S. et al., Endocrinology, 118, 2262-2267, 1986), bovine pituitary gland (Murthy, A. S. N. et al., J. Biol. Chem., 261, 1815-1822, 1986; Eipper, B. A. et al., Mol. Endocrinol 1, 777-790, 1987), rat pituitary gland (Mehta, N. M. et al., Arch. Biochem. Biophys., 261, 44-54, 1988; Birtelsen, A. H. et al., Arch. Biochem. Biophys. 279, 87-96, 1990; Stoffers, D. A. et al., Proc. Natl. Acad. Sci. USA 86, 735-739, 1989), and human origin (Glauder, J. et al., Biochem. Biophys. Res. Commun., 169, 551-558, 1990).
The cDNA coding for the C-terminally amidating enzyme PAM originating from the skin of Xenopus laevis was inserted into a Baculovirus expression vector system and expressed in insect cells. When the purified enzyme thus prepared acted upon the model peptide Ala-Ile-Gly-Val-Gly-Ala-Pro-Gly prepared by adding a glycine amino acid residue to the C-terminus of a peptide corresponding to the seven amino acid residues of the C-terminus of human calcitonin (hCT), the product formed was not the initially expected amidated peptide (Ala-Ile-Gly-Val-Gly-Ala-Pro-NH.sub.2) but a peptide with a C-terminal .alpha.-hydroxyglycine residue (Ala-Ile-Gly-Val-Gly-Ala-Pro-Gly-OH) (European Patent Application No. 91810163.5).
Based on this finding, it may be presumed that the physiological C-terminal amidating reaction involves two enzymatic steps, namely the first step which is the .alpha.-hydroxylation of the C-terminal glycine of the substrate peptide catalyzed by the hitherto known C-terminally amidating enzyme (PAM) and the second step which is the amidation by cleavage of the N-C bond in the .alpha.-hydroxyglycine moiety catalyzed by a another, novel enzyme.